We study positive allosteric modulation of neuronal nicotinic acetylcholine receptors (nAChRs). These ligand-gated ion channels are the seat of nicotine addiction and are implicated in a wide range of other neurological disorders. Allosteric modulation of nAChRs is growing in importance as it becomes better understood and as novel compounds with this pharmacological profile are identified. In previous work, we elucidated the mechanism of action for the compound morantel and we determined that morantel binds at subunit interfaces distinct from those that bind agonist. As an extension of this work, we now propose to identify the molecular determinants through which the modulator binding site communicates with the agonist binding site and with the channel gate directly, and to identify the molecular determinants of morantel site specificity. These studies will employ macroscopic voltage-clamp recordings combined with mutational and chemical modification analyses. To support these primary aims, we also propose to explore further the potentiation mechanism by single-channel recordings and mathematical modeling. Our work is innovative because we are challenging the paradigm of activation of nAChRs by occupying two agonist binding sites: We are studying a previously unknown nAChR ligand binding site, and the work we propose stands to substantially strengthen the foundation for rational design of nAChR positive allosteric modulators, a drug class with possible clinical applications. PUBLIC HEALTH RELEVANCE: We study nicotinic acetylcholine receptors, the family of proteins in the brain responsible for addiction to nicotine and implicated in a variety of other neuropathologies. Using molecular biology and electrophysiology techniques in the proposed work, we will further elucidate the molecular pharmacological mechanisms by which a certain drug class (allosteric modulators) can up-regulate the function of these proteins.